Cellular and Molecular Mechanisms of Photodynamic Hypericin Therapy for Nasopharyngeal Carcinoma Cells □ S Xiaoli Wang, Yi Guo, Shu Yang, Caihong Wang, Xuping Fu, Jinling Wang, Yumin Mao, Junsong Zhang, and Yao Li State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, People’s Republic of China (Y.G., S.Y., X.F., Y.M., Y.L.); and School of Applied Chemistry and Biological, Shenzhen Polytechnic, Shenzhen, Guangdong, People’s Republic of China (X.W., C.W., J.W., J.Z.) Received March 31, 2010; accepted June 11, 2010 ABSTRACT Hypericin-mediated photodynamic therapy (HY-PDT) has become a potential treatment for tumors and nonmalignant disorders. Some studies reported that HY-PDT could lead to apoptosis in some carcinoma cells. However, the molecular mechanism of HY-PDT remains unknown. In this study, we evaluated the mo- lecular mechanisms of hypericin associated with light-emitting diode irradiation on the poorly differentiated human nasopharyn- geal carcinoma cell line CNE-2 in vitro. To comprehensively un- derstand the effects of HY-PDT on CNE-2 cells, we detected cell viability, cell cycle, apoptosis, intracellular glutathione content, and intracellular caspase (caspase-9, caspase-3, and caspase-8) activity. Furthermore, we performed genome-wide expression analysis via microarrays at different time points in response to HY-PDT, and we found that differentially expressed genes were highly enriched in the pathways related to reactive oxygen species generation, mitochondrial activity, DNA replication and repair, cell cycle/proliferation, and apoptosis. These results were consistent with our cytology test results and demonstrated that caspase- dependent apoptosis occurred after HY-PDT. Taken together, both cellular and molecular data revealed that HY-PDT could inhibit the growth of CNE-2 cells and induce their apoptosis. Introduction Photodynamic therapy (PDT) is one of the newest advance- ments in the management of different microbial, viral, fun- gal, and inflammatory disorders and a variety of cancers. Light-induced growth inhibition is used in this method. It involves the targeting of cells or tissues that have been sen- sitized to light by administration of a photosensitizing agent. One such agent is hypericin (HY; 1,3,4,6,8,13-hexahydroxy- 10,11-dimethyl-phenanthro[1,10,9,8-opqra]perylene-7,14- dione; Falk, 1999), a secondary metabolite that can be isolated from the plant Hypericum performatum, commonly known as St. Johns wort. Because of its photoactive properties and low cytotoxicity, attention has been focused on its application in PDT (Okpanyi et al., 1990; Kersten et al., 1999; Agostinis et al., 2002; Roscetti et al., 2004; Kiesslich et al., 2006). Apoptosis and necrosis are two kinds of PDT-induced cell death (Fiers et al., 1999). Which pathway is induced depends on the different properties of the photosensitizer, the type of cells, the density of population, and the experimental method. Furthermore, the method itself varies by photosen- sitizing agent concentration, light dose, and incubation time (Blank et al., 2002; Alvarez et al., 2003). Which pathway the cell takes to PDT-induced death is organelle-dependent as well. That is, plasma membrane and lysosome can lead to necrosis, whereas mitochondrial activity can lead to pro- grammed cell death, including both caspase-dependent and -independent apoptosis (Chen et al., 2000). Caspase-depen- dent apoptosis includes two pathways, the extrinsic death pathway (death receptor-dependent) and the intrinsic death pathway (mitochondria-dependent). In the past decades, mi- tochondria has played an important role in initiating and executing apoptosis in several types of cells (Green and Kro- emer, 2004; Bras et al., 2005; Zoratti et al., 2005). This work was supported by the Technology Fund of Shenzhen Bureau of Science Technology and Information [Grant 06KJP038] and was also a part of Project 30860081 supported by the National Natural Science Foundation of China. X.W. and Y.G. contributed equally to this article. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.110.168856. □ S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: PDT, photodynamic therapy; HY, hypericin; HY-PDT, hypericin-mediated photodynamic therapy; LED, light-emitting diode; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; HP, hematoporphyrin; MTT, methylthiazolyldiphenyl-tetrazolium bromide; PBS, phosphate- buffered saline; IR, inhibition rate; GSH, glutathione; MCB, monochlorobimane; FITC, fluorescein isothiocyanate; ROS, reactive oxygen species; DE, differentially expressed; FADD, FAS-associated death domain-containing protein; HSP, heat shock protein. 0022-3565/10/3343-847–853$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 334, No. 3 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 168856/3616668 JPET 334:847–853, 2010 Printed in U.S.A. 847 at Beijing Book Co Inc/PeriodicalsFudan Univ Med Ctr/Shanghai/149411 on March 6, 2011 jpet.aspetjournals.org Downloaded from DC1.html http://jpet.aspetjournals.org/content/suppl/2010/06/15/jpet.110.168856. 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